Establishment and evaluation of a PRRSV-sensitive porcine endometrial epithelial cell line by transfecting SV40 large T antigen.

BACKGROUND: PRRSV is an infectious illness causing lung injury and abortion in sows. Cells apoptosis in the interface between the endometrium and fetal placenta is a crucial factor causing abortion. Previous study confirmed PRRSV could cause apoptosis of macrophages but rarely produced an obvious change in porcine endometrial epithelial cells (PECs). Recently, PRRSV-induced abortion was attributed to fetal placental and endometrium epithelial cells (Sn+ and CD163+) apoptosis. However, the mechanism of abortion is still unrevealed because of the limit of porcine endometrium epithelial cells (PEC). The aim of this study was to establish a stable immortalized PECs lines and use it to reveal the abortion mechanism. RESULTS: In this study, highly purified primary PECs were harvested through differential digestion, and their characteristics were confirmed by CK18, ERɑ and PR staining. Cells were then immortalized by transfecting a lentiviral vector that expressed SV40 large T antigen. PECs lines were obtained after puromycin screening. Proliferation of cell line was evaluated by cell growth curve and cell cycle assays. Cell lines exhibited faster proliferation capacity than primary cells. Biological characteristics of cell line were assessed by Western blot, karyotype analysis and staining, which confirmed that the cell line retained the endometrium characteristics. Finally, PRRSV sensitivity was assessed; expression of Sn and CD163 indicated that primary PECs and cell lines were all potentially sensitive to PRRSV. PRRSV infection tests showed an obvious increase in apoptotic rate in the infected PEC cell line, which suggested its susceptibility. CONCLUSION: The newly constructed cell line is a useful tool for studying the mechanism of abortion caused by PRRSV.

[Treatment of greater trochanter fracture after total hip replacement].

OBJECTIVE: To explore the treatment of greater trochanter fracture after total hip replacement (THR). METHODS: This series consisted of 3 males and 8 females with fractures of greater trochanter after total or partial hip replacement from 2004 to 2009. The mean age at the time of fracture diagnosis was 69 years old (range: 52 - 79). Two fractures were seen on radiograph at Day 2 postoperation and 3 found during a regular follow-up within the first 7 months after primary THR. They had no symptoms. Six patients fell and five of them occurred over 2 years after THR. The average migration between trochanter and proximal femur were 12 mm (range: 3 - 38). Ten cases underwent protected weight bearing for 6 - 12 weeks and avoidance of active abduction until union was complete or there was no pain. Only one case suffered hip dislocation with a 38-mm migration and underwent operative repair of greater trochanter with suture. Hip pain, instability, range of motion and Trendlenburg gait were examined during a regular follow-up. Evaluations of fracture migration and bone healing were performed from the radiograph of AP and lateral views of hip. RESULTS: There was no patient loss. The mean follow-up period was 40 months (range: 12 - 68). All had nearly normal function and no symptoms at the latest follow-up. For 10 non-operatively treated cases, the fractures remained non-displaced and the Trendlenburg sign was negative at the final visit. Bone healing occurred in 5 patients. Fibrous union occurred in another 6 patients. CONCLUSION: The post-THR fractures of greater trochanter are usually stable and may be treated non-operatively.

[Identification and metabolism characterization of a Clostridium lituseburense strain isolated from high-altitude soil].

OBJECTIVE: We studied the physiological, biochemical properties and metabolism of Clostridium lituseburense P4-1 from soil in Namucuo. METHODS: We adopted Hungate anaerobic technique to get Strain P4-1 from soil in Namucuo. Through physiological, biochemical and phylogenetic analysis, we identified the strain P4-1. RESULTS: Cells were Gram-positive and spore-forming. It grew between 13 and 40 degrees C (optimum at 37 degrees C), between pH value 5.0 and 10.O (optimum at 7.5-8.0), and with the presence of NaCl between 0%-5%. Strain P4-1 could metabolize many carbon sources including glucose, melibiose and mannitol. Metabolites of glucose were acetate, butyrate, propionate, CO2, and little H2. Based on 16S rDNA studies, strain P4-1 was most close to Clostridium lituseburense DSM 797 (M59107) with 98.7% similarity. Strain P4-1 could degrade p-toluene sulfonate. CONCLUSION: Strain P4-1 tolerated low temperature, salt and could degrade p-toluene sulfonate. Its metabolites produced by fermentation of glucose could improve the soil micro-environment. It was significant for strain P4-1 to be utilized in the wastewater treatment.

[Isolation and identification of a methanogen from the high temperature oil reservoir water].

To explore new microbial resources in deep subsurface oil reservoirs, strain DL-7 was isolated with Hungate technology from oil reservoir water sampled from Dagang oilfield, China. Physiological and biochemical examinations showed that H2/CO2 is the unique substrate of the strain, which cannot metabolize formate, methanol, trimethylamine, acetate and other secondary alcohols. The optimum growth conditions were further identified to be 60 degrees C, pH 7.0-7.5 and 0.25% NaCl. Moreover, the strain cannot grow without yeast extract. Analysis of its 16S rRNA sequence indicated that a similarity of 99.7% presents between the strain and the model species M. marburgensis DSM2133T (X15364).

[Isolation and identification of a thermophilic anaerobic bacterium].

OBJECTIVE: To find new microbial resources from a high-temperature oil reservoir. METHODS: Strain HL-3 was isolated by Hungate Anaerobic Technique from oil reservoir water sampled from Dagang oilfield, China. Through physiological, biochemical and phylogenetic analysis, the strain HL-3 was classified. RESULTS: Cells were Gram-positive. The temperature range for growth was 40 degrees C-75 degrees C (optimum at 60 degrees C) and the pH range was 5.0-8.0 (optimum at 6.5). The isolate could grow in the presence of 0%-3.2% NaCl (optimum at 0.25%). Glucose, ribose, mannose, xylose and cellobiose could be metabolized. Metabolites of glucose were ethanol, acetate, CO2 and trace amount of propionate and butanol. The G + C content of DNA was 33.9 mol%. Based on 16S rRNA studies,strain HL-3 was most close to T. uzonensis DSM 18761T (EF530067) with 98.8% similarity and to T. sulfurigignens DSM 17917T (AF234164) with the 98.1% similarity. Strain HL-3 tolerated to high sulfite (0. 1mol/L) ions and extremely high concentration of thiosulfate (0.8 mol/L). When the concentration of thiosulfate was higher than 0.075 mol/L, the cell would generate S element granular. The presence of H2S gas was detected inside of space at the top of serum bottle. Strain HL-3 together with T. uzonensis DSM 18761T differed greatly in toleration of thiosulfate and sulfite. The toleration of strain HL-3 to thiosulfate and sulfite was most close to T. sulfurigignens DSM 17917T (AF234164). In addition, strain HL-3 to metabolite thiosulfate and sulfite was also similar with T. sulfurigignens DSM 17917T (AF234164). However, it differs largely from both of them to metabolize glucose. CONCLUSION: Therefore, strain HL-3 may be a new spieces of the Thermoanaerobacter, and the definitive classification positioning is still awaiting for further verified with the method of determination of whole-genome DNA-DNA similarity